Abstract: Poor antigenicity and immunosuppressive ability of cancers pose major hurdles to vaccine development. Also, genetic differences in immune responses to an antigen result in an antigenic profile that varies from patient to patient. Despite tremendous progress in the cancer vaccine field, an efficient and easy method for preparing and administering vaccines that are customized to each individual patient is highly desirable, but is currently lacking in clinical settings. In this grant, we propose to evaluate the efficacy of a novel membrane-based breast cancer vaccine that can be rapidly prepared in clinical settings using the patient's tumor cell membranes incorporated with glycosyl phosphatidylinositol (GPI)-anchored cytokines by protein transfer technology. To accomplish the major objectives of the current research proposal, we have generated the GPI-anchored forms of interleukins and have successfully shown that they are as functionally active as soluble forms. Murine breast cancer cells expressing membrane bound GPI-IL-12 or GPI-IL-2, are effective in inducing protective anti-tumor immunity when mice are challenged with whole cells. In this grant application, we propose to: 1) Purify and incorporate GPI-anchored cytokines by protein transfer onto isolated breast cancer membrane vesicles and test their efficacy in inducing protective antitumor immunity in mice; 2) Investigate tumor-specific T cell and antibody responses, quantify antigen-specific CD8+ T cell expansion, and characterize memory T cell phenotype in mice vaccinated with GPI-cytokine incorporated breast cancer membrane vaccines; 3) Identify immunological mechanisms correlating with antitumor immunity elicited by the breast cancer membranes decorated with GPI-cytokine adjuvants and 4) Study tumor regression and immune responses induced by vaccination with GPI-cytokine-membranes in mice with established tumors. The advantages of the proposed membrane vaccine strategy for breast cancers are: 1) most breast cancer associated tumor antigens identified are membrane associated antigens, 2) GPI- cytokines can be stably incorporated onto membrane fragments obtained from tumor tissue thus obviating the need for establishing cell lines from patients for vaccine development, 3) vaccine preparation can be accomplished in a day and can be applied to 100% of breast cancer patients from whom tumor tissues are available, 4) due to their membrane-bound nature, GPI-cytokines will stay in the vaccination site and are unlikely to cause systemic toxicity, and 5) these cytokine-decorated tumor membranes will have the ability to deliver tumor antigens to antigen-presenting cells, such as dendritic cells, and can simultaneously activate them to elicit an effective anti-tumor immunity that can overcome tumor-induced immunosuppressive environment. The successful demonstration of this vaccine modality has the potential for development of effective membrane vaccines that are easy to prepare, store and administer to the unique tumors of individual breast cancer patients under most of the clinical settings.